Regulatory sequences for regulation of gene expression in plants and other organisms, and compositions, products and methods related thereto

ABSTRACT

The invention relates to regulatory sequences that regulate gene expression in the mesocarp and/or senescent leaves of certain plants. In certain embodiments, the invention is directed to abundant and selective expression of type 3 metallothionein-like genes in the mesocarp of a variety of plants including members of the Palme family. The invention also relates to methods of generating transgenic plants and plant tissues that comprise a nucleic acid of the invention. The invention further provides products derived from transgenic plants, plant materials or plant cells of the invention. Particular applications in oil palms are discussed. The invention further relates to nucleic acid constructs of the invention in cells of organisms other than plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 10/141,773, filed May 10, 2002, which has issued as U.S. Pat.No. 7,173,120, and which claims the benefit of Malaysian PatentApplication No PI 20021165, filed Mar. 29, 2002, each of which isincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The fatty acid composition of vegetable oils determines their physicaland chemical properties and hence their applications. The wide range ofapplications of vegetable oils, 90% edible and 10% non-edible, reflectstheir fatty acid compositional diversity. Vegetable oils are a renewableresource that can serve as feedstock to produce environmental friendlyindustrial products such as lubricants, paints, detergents and bodycare. Demand for these oils in the non-food sector is likely to increasein response to the shrinking reserves of mineral oils.

Hundreds of different fatty acids have been identified and characterisedin the plant kingdom, but most of them are not available for economicuses. Genetic engineering provides the means to tap these vast resourcesby producing fatty acids of economic importance in the storage lipids ofoil crops. Achievements through breeding alone or in combination withmutagenesis, such as in the development of rapeseed oil with low contentof erucic acid (22:1) and sunflower oil with high level of oleic acid,indicate that plants can tolerate a wide variation in fatty acidcomposition of storage lipids.

Palm oil is generally extracted from the mesocarp of the oil palmfruits. Palm oil which contains about 50% saturated, 40%monounsaturated, 10% polyunsaturated fatty acids is a semi-solid fat atroom temperature. Its fatty acid composition consists of 44% palmiticacid (16:0), 5% stearic acid (18:0), 39% oleic acid (18:1) and 10%linoleic acid (18:2). Palm oil products are primarily used in the foodsector, typically as solid fat for margarine, shortening and cooking oilproduction. Non-edible or technical applications are, however,substantial and increasing. These include, for example, soap,oleochemical-production and use as an energy source for cars.

Production of novel high-value products by genetic engineering providesthe opportunity to diversify the use and to increase the economic valueof palm oil. Production of specialty oils for industrial applicationswould be a very attractive proposition for the oil palm, since it is themost productive of oil crops. Recombinant DNA technology has been usedsuccessfully to manipulate fatty acid biosynthetic pathways in a varietyof transgenic oil crops, such as rapeseed, to produce a modified oilcomposition. Reported success in raising the levels of lauric acid andstearic acid in rapeseed oil proved that both fatty acid chain lengthand the level of fatty acid unsaturation can be modified.

Genetic engineering efforts rely heavily on the availability of areliable transformation technique for achieving stable integration and aregulatory sequence for controlling expression of introduced genes. Withoil palms, there has been significant progress in the development of areliable transformation system using biolistics techniques. In addition,analysis of several plant promoters located 5′ of genes using transgenicplant systems showed that regions in the range of several hundredbasepairs to about one kilobase could produce faithful expressionpatterns of reporter genes in vivo. The availability of seed-specificpromoters that drive gene expression over the entire period of oildeposition in oil-bearing crops like rapeseed and soybean have been amajor contribution to the success in altering oil composition by geneticengineering. These promoters have ensured that most of the effects onlipid metabolism are confined to storage lipids without significantlyaffecting lipid metabolism in leaves or other tissues which canotherwise leads to deleterious agronomic effects on the transgenicplants.

Similarly for the oil palm, efforts to modify mesocarp oil compositionby genetic engineering would benefit greatly from the availability oftemporally-regulated and tissue-targeted gene promoters. Such promotersare preferably able to drive specific expression of introduced genes inthe mesocarp during the period of oil synthesis (15-20 weeks afteranthesis). In addition, promoters for selective expression of desiredgenes in other tissues of the oil palm would also be desirable.

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery of regulatorysequences that regulate gene expression in the mesocarp and/or senescentleaves of certain plants. Some aspects of the invention relate toregulatory nucleic acids that modulate expression of a type 3metallothionein-like gene, MT3-A, in the oil palm. Nucleic acidsequences of the invention may be used to regulate expression ofintroduced genes in a variety of plant species, and, for example, totarget gene expression to the mesocarp or to senescent leaves. Themesocarp often contains the majority of useful compounds, such as oilsor carbohydrates, that are found in a fruit, and therefore the abilityto target gene expression to the mesocarp is useful, for example, tointroduce desirable traits into this tissue. Exemplary desirable traitsinclude the production of oils with modified fatty acid compositions,the production of value added nutritive compounds, the production ofpharmaceutically useful polypeptides, etc. Expression of heterologousproteins in senescent leaves is advantageous, in part, because it isunlikely to affect aspects of leaf function, such as photosynthesis,that are important for plant health. Other aspects of the inventionrelate to metallothionein-like genes and their uses in transgenicplants.

In one aspect, the invention features isolated MT3-A regulatory nucleicacids and complements thereto. In certain embodiments, the MT3-Aregulatory nucleic acids comprise nucleic acids that are at least 75%identical to the nucleic acid of SEQ ID No:1. In other embodiments, theMT3-A regulatory nucleic acids comprise nucleic acids that are at least80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the nucleic acid ofSEQ ID No:1. In a further embodiment, the MT3-A regulatory nucleic acidscomprise nucleic acids that are a portion of SEQ ID No. 1, such as, forexample, 3 consecutive nucleotides, 4 consecutive nucleotides, 5, 10,15, 20, 25, 30, 35, 40, or 50 or more consecutive nucleotides, or afunctionally active assemblage of several portions of the nucleic acidof SEQ ID No: 1. In an another embodiment, the claimed nucleic acidhybridizes with at least a portion of the nucleic acid sequence providedas SEQ. ID. No. 1 or to at least a portion of the complement of thenucleic acid sequence designated as SEQ. ID. No: 1. In preferredembodiments, claimed MT3-A regulatory nucleic acids retain the abilityto selectively stimulate gene expression in the mesocarp. In certainembodiments, MT3-A regulatory nucleic acids selectively promote geneexpression in the mesocarp, and preferably during the period of oilsynthesis. In certain embodiments an MT3-A regulatory nucleic acidsselectively promote gene expression in the mesocarp at a time between 12and 20 weeks and preferably between 15 and 20 weeks after anthesis(w.a.a.). In further embodiments, an MT3-A regulatory nucleic acidselectively promotes gene expression in senescent leaves.

In other aspects, the invention provides probes and primers comprisingsubstantially purified oligonucleotides, which correspond to a region ofnucleotide sequence which hybridizes to at least 12 consecutivenucleotides of the sequence set forth as SEQ ID No: 1 or to thecomplement of the sequence set forth as SEQ ID Nos: 1 or naturallyoccurring mutants thereof. In certain embodiments, the probe/primerfurther includes a label group attached thereto, which is capable ofbeing detected.

The invention further provides nucleic acid constructs comprising anMT3-A regulatory nucleic acid operably linked to a recombined nucleicacid. In another aspect the invention describes vectors comprised of anMT3-A regulatory nucleic acid and/or a nucleic acid construct adescribed above. Such vectors may, for example, be designed forintroduction into plant tissues, including plant cells. In a furtheraspect, the invention relates to host cells transfected with vectors,whether prokaryotic or eukaryotic, and in certain embodiments the hostcells are plant cells. In yet another aspect the invention providestransgenic plants and/or transgenic plant tissues; materials or cellsthat comprise a heterologous form of a functional or non-functionalMT3-A promoter and/or a nucleic acid construct or vector of theinvention.

The invention further relates to methods of generating transgenic plantsand plant tissues. In certain aspects, methods of the invention comprisecontacting a plant, plant tissue and/or plant cell with a vectorcomprising an MT3-A regulatory nucleic acid. Such methods may furtherinvolve regenerating one or more whole plants from transformed plant(s),plant tissue or plant cells.

In a further embodiment the invention provides transgenic plants, planttissues and plant cells comprising an MT3-A regulatory nucleic acid. Incertain embodiments, the MT3-A regulatory nucleic acid is inserted inthe plant genome at a position different from the position of anyendogenous MT3-A regulatory nucleic acids. In many embodiments, theMT3-A regulatory nucleic acid is adjacent to a recombined nucleic acid.

In other aspects the invention provides transgenic palms having arecombined nucleic acid selectively expressed in the mesocarp. Infurther aspects, the invention provides transgenic palms having arecombined nucleic acid selectively expressed in senescent leaves.

In additional aspects, the invention provides products derived fromtransgenic plants, plant materials or plant cells of the invention. Suchproducts include a wide range of materials that may generally beproduced by biologically catalyzed processes. Such products include forexample, but are not limited to, oils, polymers such aspolyhydroxybutyrates; vitamins such as carotenoids or tocopherols,celluloses, hemicelluloses etc., polypeptides, particularly polypeptideshaving pharmaceutical utility and crude preparations containing one ormore of the above and a substantial amount of plant material. In certainembodiments, the plant products of the invention are derived, at leastin part, from the mesocarp and/or senescent leaves. In furtherembodiments, the plant products of the invention are not naturallyoccurring in the host plant but are produced, at least in part, throughthe action of a gene product whose production is directed, in part or infull, by an MT3-A regulatory nucleic acid. In additional embodiments,the plant products of the invention are naturally occurring in the hostplant but in substantially different quantities. In many embodiments,plant products of the invention will contain detectable amounts ofnucleic acid comprising an MT3-A regulatory nucleic acid. In anotheraspect, the invention provides methods for determining whether a plant,plant tissue, plant cell, plant material or plant product comprises atransgenic MT3-A regulatory nucleic acid, comprising detecting thepresence of such a nucleic acid in a sample.

In another aspect, the invention features compositions that modulate anMT3-A regulatory nucleic acid, comprising molecules that modulate(agonize or antagonize) transcription from an MT3-A regulatory nucleicacid, thereby activating, increasing or suppressing the expression levelof a gene under the control of the MT3-A regulatory nucleic acid.Particularly preferred molecules for use as such modulating compositionsare selected from the group consisting of: proteins, peptides,peptidomimetics, metals, other small molecules (e.g. carbohydrates,lipids, plant hormones, or other small organic molecules) or nucleicacids (e.g. sense, antisense, ribozyme and triplex nucleic acidconstructs).

In yet another aspect, the invention provides assays for screening testcompounds to identify molecules that modulate (agonize or antagonize)transcription from an MT3-A regulatory-nucleic acid, thereby activating,increasing or suppressing the expression level of a gene under thecontrol of the regulatory nucleic acid. In one exemplary embodiment, theassay is essentially comprised of the steps of: (i) combining a testcompound with a functional reporter construct comprised of a geneencoding a reporter molecule (e.g., luciferase or GFP) under the controlof an MT3-A promoter or regulatory sequence; and (ii) detecting thelevel of expression of the reporter gene, wherein a statisticallysignificant change in the level of expression (relative to expression inthe absence of the test compound) indicates that the test compoundmodulates (agonizes or antagonizes) transcription from an MT3-Aregulatory nucleic acid.

In a further aspect, the invention provides methods of generatingtransgenic plants comprising a heterologous nucleic acid encoding ametallothionein or a nucleic acid encoding a metallothionein that isexpressed from a heterologous promoter. In one embodiment, nucleic acidsencoding a metallothionein comprise nucleic acids that are at least 75%identical to one or more of the nucleic acids of SEQ ID Nos:2-4. Inother embodiments, nucleic acids encoding a metallothionein comprisenucleic acids that are at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the nucleic acid of SEQ ID Nos:2-4. In a furtherembodiment, the nucleic acids encoding a metallothionein comprisenucleic acids that are a portion, or a functionally active assemblage ofseveral portions of, the nucleic acid of SEQ ID No:2-4. In preferredembodiments, nucleic acids encoding a metallothionein retain the abilityto interact with metals, and particularly transitional metals and/or d10metals.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 a provides the nucleotide and deduced amino acid sequences ofpOPSN6 (cDNA sequence of MT3-A). The ORF is capitalised and the UTRs arein lower case. The consensus sequences for translation start andpolyadenylation signal are doubly underlined. The amino acids are shownin single letter codes. (SEQ ID NO: 2)

FIG. 1 b provides the nucleotide sequence of the cloned 5′-RACE product.The 5′ extension to the original cDNA sequence of pOPSN6 and thecomplementary sequence to the nested primer used in the secondary PCRreaction are bold and underlined.

FIG. 2 provides the comparison of the deduced amino acid sequence ofpOPSN6 with several type 1, type 2 and type 3 metallothionein-likegenes. Dots have been introduced to optimise alignment. Dark shadedareas represent identical amino acids and light shaded areas representsimilar amino acids. Sequences from GenBank and Swiss Prot databases aregrouped to reflect the arrangement of Cys-residues.

-   -   Type 1 sequences are pea (Pisum sativum P20830) (SEQ. ID. NO:        16), maize (Zen mays P30571) (SEQ. ID. NO: 17), clover (Triflium        repens P43399) (SEQ. ID. NO: 18), and rice (Orzya sativa Q40633)        (SEQ. ID. NO: 19).    -   Type 2 sequences are rice (Oryza saliva D15602), (SEQ. ID. NO:        20), arab (Arabidopsis thaliana P25860) (SEQ. ID. NO: 21), kiwi        fruit (Actinidia delicosa P43390) (SEQ. ID. NO: 22) and castor        bean (Ricinus communis P30564) (SEQ. ID. NO: 23).    -   Type 3 sequences are kiwi fruit (Actinidia delicosa P43389)        (SEQ. ID. NO: 24), banana (Musa acuminata 40256) (SEQ. ID.        NO: 25) and papaya (Carica papaya Q96386) (SEQ. ID. NO: 26).

FIG. 3 a shows the alignment between deduced amino acid sequences ofpOPSN6 (MT3-A) and pOPSN7 (MT3-B).

FIG. 3 b is a table summarising percentages of identity at thenucleotide and amino acid levels between sequences of pOPSN6 and pOPSN7(cDNA sequence of MT3-B).

FIG. 4 provides the result of Southern analysis for gene copy numberdetermination of MT3-A and MT3-B. Ten micrograms of oil palm (Elaeisguineensis) genomic DNA digested with Eco RI, Hind III or Xba 1, wasseparated on 0.9% agarose gel, transferred to nylon membrane and probedwith gene-specific probes based on 3′UTR of MT3-A and MT3-B gene.

FIG. 5 gives the results of Northern analysis of oil palm MT3-A geneshowing its expression pattern in young leaves, germinated seedlings,kernel at 12 w.a.a and mesocarp tissues at different stages (5, 12, 15,17 and 20 w.a.a) of development. Northern blot containing 2 μg poly (A)⁺RNA from various oil palm (E. guineensis) tissues was hybidised with³²P-labelled probe prepared using entire insert of MT3-A cDNA. Anethidium bromide stained gel (B) was included to show approximatelyequal loading of poly (A)⁺ RNA samples.

FIG. 6 compares the expression pattern of MT3-A and MT3-B genes in youngand senescent leaves, roots, and kernel at 3 different stages (10, 12and 15 w.a.a) and mesocarp at 4 different stages (8, 12, 15 and 17w.a.a) of development. Northern blot containing 20 μg total RNA fromvarious oil palm (E. guineensis) tissues was hybridised with³²P-labelled gene-specific probe from 3′-untranslated regions of MT3-Aand MT3-B genes (as shown). An ethidium bromide stained gel (C) wasincluded to show approximately equal loading of RNA samples.

FIG. 7 gives the results of Northern analysis of oil palm stearoyl-ACPdesaturase genes showing their expression patterns in young leaves,germinated seedlings, kernel at 3 different stages (10, 12 and 14 w.a.a)and mesocarp at 6 different stages (8, 10, 12, 15, 17 and 20 w.a.a) ofdevelopment. Northern blots containing 2 μg poly (A)⁺ RNA from variousoil palm (E. guineensis) tissues (lanes 2-12) were hybridised with³²P-labelled gene-specific probe from 3′-untranslated regions of oilpalm stearoyl-ACP genes, SAD1 and SAD2. Lane 1 represents E. oleiferamesocarp at 15 w.a.a. An ethidium bromide stained gel (C) was includedto show approximately equal loading of RNA samples.

FIG. 8A shows the products of the primary PCR reaction using primersGSPI and AP 1. Lanes 1, 2, 3 and 4 are the products obtained using 1 μlaliquots of the Dra I, Eco RV, Pvu II and Stu I GenomeWalker libraries,respectively. Lane 5 is the product from control library provided withthe GenomeWalker Kit.

FIG. 8B shows the amplified DNA fragment obtained from the secondary PCRreaction using primers GSP2 and AP2. Only the primary PCR product fromDra I library was used in the reaction. The band was subsequentlypurified from the agarose gel and cloned into the PCR II TOPO vector forsequencing. Lanes 1 and 2 are the secondary PCR products from Dra Ilibrary and control library, respectively.

FIG. 9 provides the nucleotide sequence of pMT3A-P1a (the promotersequence of MT3-A). The putative ethylene responsive element(ERE-reverse), TATA box, the adenine at the 5′ end of the 5′ RACEproduct (likely transcription start site) and the ATG codon for start oftranslation are bold and underlined. (SEQ ID NO: 1).

FIG. 10 shows DNA sequence alignment of pMT3A-Pla (3′-end of nucleotide1000) (SEQ. ID. NO: 27) and the sequence of the 5′ RACE product(nucleotides 1-130 of SEQ. ID. NO: 5) clearly showing 100% homologywithin the overlapping region (SEQ. ID. NO: 28).

FIG. 11 a shows the result of restriction analysis of the chimerictransformation vectors MT3AP-EGFP containing 986 bp MT3-A promotersequence and GFP as reporter gene. Lane 1: pEGFP vector uncut, Lane 2:MT3AP-EGFP uncut, Lane 3: MT3AP-EGFP digested with Hind III and Pst I

FIG. 11 b shows the result of restriction analysis of the chimerictransformation vectors MT3AP-GUS containing 986 bp MT3-A promotersequence and GUS as reporter gene. Digestion was performed with Hind IIIand Xba I. Lane 1 shows digested products of pBI221. Lanes 2 and 3 showdigested products of MT3AP-GUS.

FIG. 12 is a table showing the result of optimising the helium pressurefor bombardment for, obtaining GFP expression in mesocarp and controlleaf tissue. The optimisation was performed using plasmid HBTIαcontaining CAMV 35 S enhancer fused to the basal promoter of maizeC4PPDK as described by Sheen, J (1993), [“Protenin phosphatase activityis required for light-inducible gene expression in maize”. EMBO J 12(9):3497-3505]. In HBT1α, the α represents SGFP-nos.

FIG. 13 shows expression of GFP in mesocarp tissue slices bombarded withthe plasmid MT3AP-EGFP containing the 986 bp MT3-A promoter sequence. Noexpression was observed when MT3AP-EGFP was used to bombard the controlleaf tissues.

FIG. 14 shows the results of histochemical assay to compare expressionof GUS between mesocarp and leaf tissues bombarded with the plasmidpBI221 (containing constitutive CaMV 35S promoter) and tissues bombardedwith MT3AP-GUS (containing 986 bp oil palm MT3-A promoter sequence). Thearrows point to examples of GUS spots on the bombarded tissues.

FIG. 15 shows the coding sequence for MT3-B (SEQ ID NO: 3)

FIG. 16 shows the genomic sequence for MT3-B (SEQ ID NO: 4)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

The term “fruit” as used herein is intended to encompass any object orobjects that are produced by a plant in response to a fertilizationevent, whether a self- or non-self fertilization, and whether or not theresulting fruit is sterile or non-sterile. For example, both an appleand the seeds of the apple should be viewed as “fruit” herein. Asanother example, seedless fruits such as grapes and tangerines arefruit. “Fruit” are not limited to edible objects. For example, thepoisonous berries of the yew are “fruit” as are any fruits and nutsproduced by palms. Other exemplary “fruit” include peanuts, tomatoes,corn, bananas, wheat berries, pears, etc.

“Host cells” or “recombinant host cells” or “recombinant cells” areterms used interchangeably herein. It is understood that such termsrefer not only to the particular subject cell but to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology andidentity can each be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When an equivalentposition in the compared sequences is occupied by the same base or aminoacid, then the molecules are identical at that position; when theequivalent site occupied by the same or a similar amino acid residue(e.g., similar in steric and/or electronic nature), then the moleculescan be referred to as homologous (similar) at that position. Expressionas a percentage of homology/similarity or identity refers to a functionof the number of identical or similar amino acids at positions shared bythe compared sequences. A sequence which is “unrelated” or“non-homologous” shares less than 30% identity, though preferably lessthan 25% identity with a sequence of the present invention. In comparingtwo sequences, the absence of residues (amino acids or nucleic acids) orpresence of extra residues also decreases the identity andhomology/similarity.

The term “homology” describes a mathematically based comparison ofsequence similarities which is used to identify genes or proteins withsimilar functions or motifs. The nucleic acid and protein sequences ofthe present invention may be used as a “query sequence” to perform asearch against public databases to, for example, identify other familymembers, related sequences or homologs. Such searches can be performedusing the NBLAST and XBLAST programs (version 2.0) of Altschul, et al.(1990) [J. Mol. Biol. 215:403-10] BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain ammo acid sequences homologousto protein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al. (1997) Nucleic Acids Res. 25 (17)3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters or therespective programs (e.g., XBLAST and BLAST) can be used.

As used herein, “identity” means the percentage of identical nucleotideor amino acid residues at corresponding positions in two or moresequences when the sequences are aligned to maximize sequence matching,i.e., taking into account gaps and insertions. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux, J., et al.,Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA(Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) andAltschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST Xprogram is publicly available from NCBI and other sources (BLAST Manual,Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., etal., J. Mol. Biol. 215: 403-410 (1990). The well known Smith Watermanalgorithm may also be used to determine identity.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA, or RNA, refers to molecules in a form which does not occur innature. Moreover, an “isolated nucleic acid” is meant to include nucleicacid fragments which are not naturally occurring as fragments and wouldnot be found in the natural state.

The terms “lipid metabolism” and “fatty acid metabolism” are usedinterchangeably to refer to the set or a subset of biochemical reactionsthat are involved in the biosynthesis of fatty acids and/or lipids.

The term “mesocarp” refers to the middle layer of the pericarp. Thepericarp is the outer wall of a fruit. The mesocarp is often fleshy orfibrous.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include analogs of eitherRNA or DNA made from nucleotide analogs, and, as applicable to theembodiment being described, single-stranded (such as sense or antisense)and double-stranded polynucleotides. Nucleotide analogs may havemodified base moieties, modified sugar moieties, modified phosphatemoieties, or a combination thereof. For example a nucleotide analog maybe a peptide nucleic acid or a nucleic acid having a transition metalsubstituted from the phosphate in the backbone.

A “nucleic acid construct” is a nucleic acid comprising a first nucleicacid and a second nucleic acid, wherein the first nucleic acid isadjacent to the second nucleic acid, and wherein the first and secondnucleic acids are not adjacent in a naturally occurring genome.

The terms “oil” and “lipid” are used interchangeably herein to refer toany material or mixture of materials that is primarily composed of oneor more highly hydrophobic substances such as fatty acids or true fats(e.g. esters of fatty acids and glycerol); lipids (eg. phospholipids,cerebrosides, waxes); and lipoproteins.

The term “operably linked” is used herein to refer to the relationshipbetween a regulatory sequence and a gene. If the regulatory sequence ispositioned relative to the gene such that the regulatory sequence isable to exert a measurable effect on the amount of gene productproduced, then the regulatory sequence is operably linked to the gene.

A “plant cell” is a cell derived, directly or indirectly, from a plantor a portion of a plant. This term includes plant cell lines that havebeen propagated through multiple generations. A plant cell is alsointended to include cells as present in a plant or plant material.

“Plant material” refers to any material derived from a plant, includingfor example, but not limited to, fruits, seeds, bark, oils, starches,saps, pollen, petals, sepals, roots, and fragmentary portions thereof.Plant material also includes, for example, plant cells and plant tissue.

“Plant tissue” refers to any portion of a plant that includes aplurality of cells (living or dead) and some extracellular matrix. Planttissues include, for example, but are not limited to, seeds, fruits,leaves, roots, endosperm, xylem, phloem, etc.

A “recombined nucleic acid” is any nucleic acid that has been placedadjacent to a second nucleic acid by recombinant DNA techniques. A“recombined nucleic acid” also includes any nucleic acid that has beenplaced next to a second nucleic acid by a laboratory genetic techniquesuch as, for example, transformation and integration, transposon hoppingor viral insertion. In general, a recombined nucleic acid is notnaturally located adjacent to the second nucleic acid.

A “regulatory element”, also termed herein “regulatory sequence” isintended to include elements which are capable of modulatingtranscription from a core promoter and include elements such asenhancers and silencers. The term “enhancer”, also referred to herein as“enhancer element”, is intended to include regulatory elements capableof increasing, stimulating, or enhancing transcription from a basicpromoter. The term “silencer”, also referred to herein as “silencerelement” is intended to include regulatory elements capable ofdecreasing, inhibiting, or repressing transcription from a basicpromoter. Regulatory elements can also be present in genes other than in5′ flanking sequences. Thus, it is possible to have regulatory elementslocated in introns, exons, coding regions, 3′ flanking sequences, etc.

A “reporter gene” is any gene encoding a gene product such as a protein(reporter protein) or mRNA (reporter mRNA) which can readily bedetected. Reporter proteins may include, but are not limited tofluorescent proteins (eg. Green Fluorescent Protein, Red FluorescentProtein), chromogenic enzymes (eg. beta-galactosidase, alkalinephosphatase, beta-glucuronidase) and fluorogenic enzymes (eg.luciferase).

The term “selectively” as used herein in reference to gene expression isintended to mean that expression of the gene is higher, and preferably2, 5, or 10 times higher, in a particular tissue or cell type than theaveraged level of expression measured in other tissues or cell types(eg. as compared to expression level measured in a whole planthomogenate). For example, the MT3-A gene is selectively expressed in themesocarp because expression of this gene, as measured by Northern blot,is substantially higher in the mesocarp than in other tissues measured.The MT3-A gene is also selectively expressed in senescent leaves.

“Small molecule” as used herein, is meant to refer to a compound, whichhas a molecular weight of less than about 5 kD and most preferably lessthan about 2.5 kD. Small molecules can be nucleic acids, peptides,polypeptides, peptidomimetics, carbohydrates, lipids or other organic(carbon containing) or inorganic molecules. Most plant hormones, such asgiberellins, auxins, abscisic acids, cytokinins etc., are also smallmolecules.

As used herein, the term “specifically hybridizes” refers to the abilityof a nucleic acid probe/primer of the invention to hybridize to at least12, 15, 20, 25, 30, 35, 40, 45, 50 or 100 consecutive nucleotides of atarget gene sequence, or a sequence complementary thereto, or naturallyoccurring mutants thereof, such that it has less than 15%, preferablyless than 10%, and more preferably less than 5% background hybridizationto a cellular nucleic acid (e.g., mRNA or genomic DNA) other than thetarget gene. A variety of hybridization conditions may be used to detectspecific hybridization, and the stringency is determined primarily bythe wash stage of the hybridization assay. Generally high temperaturesand low salt concentrations give high stringency, while low temperaturesand high salt concentrations give low stringency. Low stringencyhybridization is achieved by washing in, for example, about 2.0×SSC at50° C., and high stringency is achieved with about 0.2×SSC at 50° C.Further descriptions of stringency are provided below.

As used herein, the term “regulatory nucleic acid” refers to a nucleicacid that activates and/or regulates expression of a selected DNAsequence when such a DNA sequence is operably linked to the regulatorynucleic acid. The term “5′ flanking sequence” can include regulatorynucleic acids, but is intended to refer more generally to any nucleicacid sequence located upstream of the transcription initiation site.Thus, a “5′ flanking sequence” of an MT3-A gene is intended to includeany nucleic acid sequence located upstream of the transcriptioninitiation site and is not required to have any transcriptionalactivity. The term “core promoter” as used herein is intended to referto the minimal regulatory nucleic acid that is capable of initiatingtranscription of a selected DNA sequence to which it is operably linked.The term “core promoter” is intended to represent a promoter elementproviding basal transcription. A core promoter frequently consists of aTATA box or TATA-like box and complexes with an RNA polymerase. Aregulatory may or may not include a core promoter.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Preferred vectors are thosecapable of autonomous replication and/expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. Expression vectors of utility in recombinant DNAtechniques are often in the form of “plasmids” which refer to circulardouble stranded DNA loops which, in their vector form are not bound tothe chromosome. Viral vectors are also frequently used as expressionvectors.

Regulatory Nucleic Acids

One aspect of the invention pertains to isolated nucleic acids selectedfrom the group consisting of a nucleic acid having SEQ ID No: 1,functional variants and fragments thereof, MT3-A regulatory elements,equivalents to any of these nucleic acids, and complements to any ofthese nucleic acids. The invention also pertains to nucleic acidscapable of hybridizing to the nucleic acid sequence shown SEQ ID No: 1or a complement thereof. Also within the scope of the invention arenucleic acids which are homologous, e.g., 80%, 85%, 90%, 95%, 98%, 99%or 100% identical to any of the above-recited nucleic acids.Accordingly, the invention provides nucleic acids which are capable offunctioning as a promoter, nucleic acids which are capable offunctioning as a regulatory element, as well as nucleic acids which donot necessarily function as either. A “functionally active” nucleic acidas used herein with respect to transcriptional regulatory sequences is anucleic acid that is capable of modulating transcription of a gene towhich it is operably linked. Functionally active nucleic acids may beone of the nucleic acids above, including nucleic acid fragments. Thus,a “functionally active” nucleic acid is intended to include nucleicacids capable of functioning as a promoter or as a regulatory element inappropriate conditions. A functionally active fragment of the nucleicacid can be a portion of the nucleic acid which providesmesocarp-selective expression and/or senescent leaf-selectiveexpression. A preferred portion of the nucleic acid provides tissuespecific expression substantially similar to the tissue distributionand/or temporal pattern of MT3-A. It is also understood that promotersare often modular in structure, with regulatory elements that greatlyaffect function and other inter-element sequences with little importanceexcept, in certain instances, to proved the appropriate spacing betweenregulatory elements. Accordingly, a functionally active nucleic acid mayinclude one or more portions of one of the above nucleic acidsassembled, but not necessarily contiguously, to provide a nucleic acidhaving regulatory activity. The term equivalent of a nucleic acid isunderstood to include nucleic acids which differ by one or morenucleotide substitutions, additions or deletions from the nucleic acidand which has a similar activity as the nucleic acid.

Preferred nucleic acids of the invention are from the upstream regionsof plant genes encoding metallothioneins. A particularly preferrednucleic acid of the invention is an oil palm nucleic acid, such as anucleic acid having SEQ ID No: 1 or a portion thereof. Regardless ofspecies, particularly preferred nucleic acids are at least 80%, 85% 90%,95% or 99% similar or identical to the nucleic acids shown in SEQ ID No:1.

Another aspect of the invention provides a nucleic acid which hybridizesto the nucleic acid shown in SEQ ID No: 1 or complement thereof.Appropriate stringency conditions which promote DNA hybridization, forexample, 6× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2×SSC at 50° C., are known to those skilled in theart or can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. Inaddition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In a preferred embodiment, a nucleic acidof the present invention will bind to SEQ ID No. 1 under moderatelystringent conditions, for example at about 2.0 times SSC and about 40°C. In a particularly preferred embodiment, a nucleic acid of the presentinvention will bind to SEQ ID No: 1 or complement thereof under highstringency conditions. In certain embodiments the hybridizing nucleicacid is at least 20, 25, 30, 50, 100 or 250 nucleotides in length.Hybridization can be used to isolate nucleic acids of the inventioncorresponding to MT3-A promoters and regulatory regions from variousplant species. A comparison of these nucleic acids should be indicativeof regions involved in the regulation of expression of the MT3-A gene,since these regions are expected to be conserved among various species.

Other nucleic acids of the invention are nucleic acids corresponding toone or more discrete regulatory elements, such as enhancers andsilencers. Preferred nucleic acids include an ERE box and/or TATA box asidentified in FIG. 9.

Any nucleic acid fragment of the invention can be prepared according tomethods well known in the art and described, e.g., in Sambrook, J.Fritsch, E. F., and Maniatis, T. (1989 Molecular cloning a laboratorymanual (second edition). New York: Cold Spring Harbor Laboratory Press)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). For example, discrete fragments of thepromoter can be prepared and cloned using restriction enzymes.Alternatively, discrete fragments can be prepared using the PolymeraseChain Reaction (PCR) using primers having an appropriate sequence, suchas a sequence in SEQ ID NO: 1 or complement thereof.

In yet another embodiment of the invention, the isolated nucleic acidcomprises a nucleic acid sequence of SEQ ID No: 1 or portion thereofwhich has been modified, e.g., by adding, deleting, or substituting oneor more nucleic acid residues. Such modifications can modulate thetranscriptional activity of the MT3-A regulatory nucleic acid. Forexample, a modification can increase or decrease the activity of apromoter or regulatory element. A modification can also affect thetissue specificity of a promoter or regulatory element. Thus, forexample, an MT3-A regulatory nucleic acid may be modified to stimulatetranscription in only one of the tissues in which it is normallyexpressed, such as the mesocarp or senescing leaves.

Desired modifications of an MT3-A promoter or regulatory element can begenerated according to methods that, in view of this specification, arewell known in the art, such as by site-directed or random mutagenesis.The activity of the modified promoter or regulatory element can then betested, e.g., by cloning the modified promoter upstream of a reportergene, transfecting the construct into plant cells, such as plantmesocarp or leaves or a cell suspension, and measuring of the level ofexpression of the reporter construct. Such methods are further describedin the examples. The activity of the modified promoter or regulatoryelement can also be analyzed in vivo in transgenic plants. It is alsopossible to create libraries of modified fragments which can be screenedusing a functional assay, in which, for example, only modified promotersor regulatory elements having the desired activity are selected. Theseassays can be based, e.g., on the use of reporter genes providingresistance to specific selection agents such as herbicides orantibiotics, e.g. kanamycin. Selection of cells having a reporterconstruct containing a promoter or regulatory element having the desiredmodification can be isolated by culture in the presence of the selectionagent.

Also within the scope of the invention are nucleic acid constructscomprising an MT3-A regulatory nucleic acid operably linked to arecombined nucleic acid to be transcribed. The MT3-A regulatory nucleicacid can be, e.g., any of the above-described fragments or variants, andis preferably functional. The MT3-A regulatory nucleic acid can also bea combination of several fragments or regulatory elements having asequence from SEQ ID No: 1 or modified form thereof, as well asmultimers of one or more of these fragments or regulatory elements ormodified form thereof. The promoter can also contain regulatory elementsderived from other genes.

In many embodiments, the recombined nucleic acid to be transcribedencodes a protein or peptide. The protein can be any protein useful inproducing desired qualities or products in the target tissue (eg.mesocarp or senescent leaves). In certain embodiments, the protein isinvolved in fatty acid and/or lipid metabolism, such as: ACCases (EC6.4.1.2), such as homomeric acetyl-CoA carboxylase, heteromericacetyl-CoA carboxylase subunits, acyl carrier proteins, malonyl-CoA:ACPtransacylases (EC 2.3.1.39), ketoacyl-ACP synthases (KAS) (EC 2.3.1.41),such as KAS I, KAS II, KAS III, ketoacyl-ACP reductases (EC 1.1.1.100),3-hydroxyacyl-ACP dehydrases (EC 4.2.1.17), enoyl-ACP reductases (EC1.3.1.44), stearoyl-ACP desaturases (EC 1.14.99.6), acyl-ACP desaturasesother than stearoyl-ACP desaturases, acyl-ACP thioesterase (EC3.1.2.14), FatA, FatB, glycerol-3-phosphate acyltransferases (EC2.3.1.15), 1-acyl-sn-glycerol-3-phosphate acyltransferases (EC2.3.1.51), plastidial cytidine-5′-diphosphate-diacylglycerol synthases(EC 2.7.7.41), plastidial phosphatidylglycerophosphate synthases (EC2.7.8.5), plastidial phosphatidylglycerol-3-phosphate phosphatases (EC3.1.3.27), phosphatidylglycerol desaturases (palmitate specific)(EC1.14.99.-) (FAD4), plastidial oleate desaturases (FAD6) (EC 1.14.99.-),plastidial linoleate desaturases (FAD7/FAD8)(EC 1.14.99.-), plastidialphosphatidic acid phosphatases (EC 3.1.3.4),monogalactosyldiacylglycerol synthases (EC 2.4.1.46),monogalactosyldiacylglycerol desaturases (palmitate-specific), (EC1.14.99.-) (FAD5), digalactosyldiacylglycerol synthases (EC 2.4.1.184)(DGD1), sulfolipid biosynthesis proteins, long-chain acyl-CoAsynthetases (EC 6.2.1.3), ER glycerol-3-phosphate acyltransferases (EC2.3.1.15), ER 1-acyl-sn-glycerol-3-phosphate acyltransferases (EC2.3.1.51) (eg. “Cocos nucifera” type and “Brassica napus” type), ERphosphatidic acid phosphatases (EC 3.1.3.4), diacylglycerolcholinephosphotransferasesg (EC 2.7.8.2), ER oleate desaturases (FAD2)(EC 1.14.99.-), ER linoleate desaturases (FAD3) (EC 1.14.99.-), ERcytidine-5′-diphosphate-diacylglycerol synthases (EC 2.7.7.41), ERphosphatidylglycerophosphate synthases (EC 2.7.8.5), ERphosphatidylglycerol-3-phosphate phosphatases (EC 3.1.3.27),phosphatidylinositol synthases (EC 2.7.8.11), diacylglycerolacyltransferases (EC 2.3.1.20), delta-8 sphingolipid desaturases, oleate12-hydroxylases, bifunctional oleate 12-hydroxylase:desaturases, delta12 fatty acid acetylenases, delta 12 fatty acid epoxygenases,diacylglycerol kinases (EC 2.7.1.107), cholinephosphatecytidylyltransferases (EC 2.7.7.15), choline kinases (EC 2.7.1.32),phospholipase C (EC 3.1.4.11), phospholipase D (EC 3.1.4.4),phosphatidylserine synthases, phosphatidylserine decarboxylases (EC4.1.1.65), phosphatidylinositol-3-kinases (EC 2.7.1.137),phosphatidylinositol-4-kinases (EC 2.7.1.67), ketoacyl-CoA synthases(KCS), beta-ketoacyl reductases (involved in wax biosynthesis), waxsynthases, oleosins, oleosin-like proteins, 3-ketoacyl-CoA thiolases (EC2.3.1.16), acyl-CoA oxidases (EC 1.3.3.6) and acyl-CoA dehydrogenases(EC 1.3.99.2).

In general, crude palm oil contains a few minor components of high valueincluding lycopene (a carotenoid), tocopherols and tocotrienols (vitaminE), sterols and squalene. In certain aspects, the recombined nucleicacid to be transcribed encodes a protein involved in the biosynthesis ofone or more of these compounds. Exemplary proteins include:Hydroxymethylglutaryl-CoA reductase (NADPH)(EC 1.1.1.34); Mevalonatekinase (EC 2.7.1.36); Phosphomevalonate kinase (EC 2.7.4.2);Diphosphomevalonate decarboxylase (EC 4.1.1.33); Geranyltranstransferase(EC 2.5.1.10); Farnesyltaanstransferase (EC 2.5.1.29);Dimethylallyltranstransferase (EC 2.5.1.1); Isopentenyl-diphosphatedelta-isomerase (EC 5.3.3.2); Chrysanthemyl pyrophosphate synthase;s-linalool synthase; Geranylgeranyl-diphosphategeranylgeranyltransferase (EC 2.5.1.32); Phytoene synthase; Phytoenedesaturase; Lycopene cyclase; Carotene 7,8-desaturase (EC 1.14.99.30);b-carotene hydroxylase; gamma-tocopherol methyl transferase;p-hydroxyphenylpyruvate dioxygenase; Squalene synthetase (EC 2.5.1.21);Squalene monooxygenase (EC 1.14.99.7); Lanosterol synthase (EC5.4.99.7); Cycloartenol synthase (EC 5.4.99.8); Lathosterol oxidase (EC1.3.3.2); Cholestenol delta-isomerase (EC 5.3.3.5); 7-Dehydrocholesterolreductase (EC 1.3.1.21). In certain embodiments, expression of one ormore of the above enzymes may enhance production of high valuebiochemicals.

In other embodiments, the protein to be expressed is useful wheningested by humans; for example, a variety of proteins from pathogensare effective as oral vaccines, such as hepatitis B surface antigen, andsuch proteins may be expressed in the mesocarp of fruits for ingestion.Proteins that promote synthesis of nutritionally valuable compounds,such as proteins involved in the biosynthesis of vitamins may also beexpressed to produce fruits having a higher content of such compounds.Proteins may also increase or decrease or otherwise modify theproduction of fibrous material and/or extracellular matrices so as toalter mesocarp texture and/or toughness.

In further embodiments proteins involved in the biosynthesis ofbioplastics, such, as polyhydroxybutyrates may be operably linked toregulatory sequences of the invention. Exemplary enzymes involved inbioplastic biosynthesis include polyhydroxyalkanoate synthase;polyhydroxybutyrate synthase; beta-ketothiolase; acetoacetyl-CoAreductase; phosphotransacetylase; polyhydroxybutyrate depolymerase; etc.In many embodiments, genes encoding the above proteins are bacterialgenes or modified genes derived from bacteria.

In another embodiment, the recombined nucleic acid is transcribed intoan RNA having an activity, such as an antisense, an RNAi, or a ribozyme.Expression of such nucleic acids can be used, e.g., to reduce or inhibittranslation of a MRNA into a specific protein or to reducetranscription.

In yet another embodiment, the nucleic acid to be regulated by an MT3-Aregulatory nucleic acid is a reporter gene. Reporter genes include anygene encoding a protein, the amount of which can be determined.Preferred reporter genes include Green Fluorescent Protein (GFP) andmany colored variants (RFP, YFP, CFP, BFP) and other fluorescentproteins, the luciferase gene, the beta-galactosidase gene (LacZ), thechloramphenicol acetyl transferase (CAT) gene, the beta-glucuronidasegene (GUS) or any gene encoding a protein providing resistance to aspecific chemical.

Probes, Primers and Detection Methods

Moreover, the MT3-A regulatory nucleic acid sequences provide for thegeneration of probes and primers which can be used, e.g. to identifyplant materials derived from a transgenic plant of the invention. Suchmethods are highly desirable for monitoring the presence of geneticallymodified plant materials in foods and other substances. The presentinvention also provides a probe/primer comprising a substantiallypurified oligonucleotide, which oligonucleotide comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast approximately 6, 8, 10 or 12, preferably about 25, 30, 40, 50 or75 consecutive nucleotides of SEQ ID No: 1.

In certain embodiments, the probe further comprises a label attachedthereto, which is capable of being detected, e.g. the label group isselected from amongst radioisotopes, fluorescent compounds, enzymes, andenzyme co-factors.

In certain embodiments, probes and primers that hybridize to an MT3-Aregulatory nucleic acid may, for example, be used to detect the presenceof materials derived from a transgenic plant or plant material thatcontains an MT3-A regulatory nucleic acid. In certain instances, it willbe desirable to perform the detection by PCR, in which case two primerswill typically be used. It is preferable to select primers that willdistinguish between a transgenic version of an MT3-A regulatory nucleicacid and an endogenous version. Such primers may be selected todistinguish by the size of fragment produced and/or by a failure toefficiently amplify the endogenous form. Primers may also be selected todetect other parts of a transgenic nucleic acid that are external to theMT3-A regulatory region. Probes and primers may be used in otherdetection schemes, such as Southern and Northern blots. In addition,many methodologies involve a global, non-specific amplification ofnucleic acids in a sample, followed by probing with a desired probe, andoften the desired probe will be part of an array of probes, such as amicorarray. In view of this specification, other methods for detectionof transgenic nucleic acids that include an MT3-A regulatory nucleicacid will be apparent to one of skill in the, and such methods areintended to be encompassed hereby.

Plants and Plant Cells

The invention provides plants, plant cells, plant tissue and plantmaterials that comprise a nucleic acid of the invention, and preferablya nucleic acid construct having a regulatory nucleic acid operablylinked to a recombined nucleic acid.

In certain embodiments, regulatory nucleic acids of the invention areuseful for directing mesocarp-selective expression in a wide range ofplants (and related cells, tissues etc.). Transgenic plants of theinvention include, but are not limited to, the palms (members of thefamily Palmae) and other plants having fruits with oil-bearing mesocarptissues, such as olives (members of the family Oleaceae) and avocados.Particularly preferred palms include date palms, coconut palms and oilproducing palms, which include but are not limited to palms selectedfrom the following genera: Acrocomia, Astrocaryum, Elaeis, Jessenia,Oenocarpus, Orbignya, and Scheelea. Preferred transgenic plants aremembers of the species Elaeis guineensis Jacq., Elaeis oleifera andhybrids thereof. Additional transgenic plants of the invention are plum,mango, tomato, berries, plum, apricot, kiwifruit, melons, cucumber,aubergine, peach, apricot, grape, peach, pear, apple, quince, papaya,and nectarine.

Methods for generating transgenic plants of the invention are, in viewof this specification, well known to those of skill in the art. Oneexemplary type of method involves the bombardment of plant cells ortissue with tungsten (or other relatively inert, dense metal) pelletsthat have been mixed with the nucleic acid to be introduced. Transformedcells may be selected, often by the presence of a selectable markerpresent in the nucleic acid. Selectable markers include antibiotic orherbicide resistance genes or chromogenic markers. A commonly usedmarker is the kanamycin resistance gene. Transformed cells may becultured to produce callus and, if desired, to regenerate whole plants.

Another exemplary type of method of plant transformation uses the soilbacterium Agrobacterium tumefaciens. The nucleic acid to be introducedinto the plant is cloned into a vector containing at least one flankingT DNA sequence. When the Agrobacterium containing this plasmid iscontacted with the appropriate plant, the DNA positioned near the T DNAsequence is inserted into the genome of cells of the plant. It iscontemplated that a variety of cloning vectors and bacterial hoststrains can be used. For example, Ti-based vectors like pGV3850 intowhich recombinant plasmids integrate before transfer to plant cells areknown as cis-type vectors. There are also Ti-based vector systems inwhich the recombinant plasmids do not integrate into the resident Tiplasmid or in which large portions of the naturally occurring Ti plasmidare deleted. These binary-type systems, Hoekema et al., Nature, Vol.303, 179 (1983), or mini-Ti plasmids, Framond et al., Biotechnology,Vol. 1, 262 (1983), have also been shown to introduce DNA into plantcells. These plasmids contain a border sequence (at least one,preferably two) flanking the gene to be introduced into plants. A markerwhich is selectable or scorable in plant cells is useful but notessential. Such plasmids are typically capable of autonomous replicationin A. tumefaciens and need not integrate into a resident Ti plasmid.Virulence functions that are useful in effecting transfer to DNA, suchas the chimeric genes of the present invention, to plant cells may beprovided in trans. Hoekema et al., Nature, Vol. 303, 179 (1983). Seealso Fraley, R. T. et al., Biotechnology, Vol. 3, 629 (1985); and Kleeet al., Biotechnology, Vol. 3, 637 (1985).

Other exemplary methods for introducing nucleic acids into plantsinclude physical methods such as electroporation, chemical methods suchas polyethylene glycol (PEG) fusion, and viral methods such as RNA viralvectors which introduce an RNA copy of a gene.

The following is an exemplary methodology for generating a transgenicoil palm. Transgenic oil palm containing the bar gene which confersresistance to the herbicide Basta™ may be produced using the biolistictechniques. Parveez (2000) [“Production of transgenic oil palm (Elaeisguineensis JACQ.) using biolistic techniques”. Molecular Biology ofWoody Plants. (Eds. S. M. Jain and S. C. Minocha). Kluwer AcademicPublishers, Vol. 2, 327-350] described the detailed protocol for theproduction of Bastam resistant transgenic oil palm, which can be used asreference for generating transgenic oil palm containing any othergene(s) of interest. Embryogenic calli derived from embryos, leafletsand roots are used in bombardment. The physical and biologicalparameters such as helium pressure, microcarrier type and explant sourcefor biolistic transformation of oil palm are optimized. Basta™ and theantibiotic hygromycin are suitable selective agents for transformedcells as they are effective at a much lower concentration (40 mg/ml)compared to other selective agents. The transgenic embryogenic callus issubsequently transferred onto fresh embryogenesis-inducing mediumfollowed by shoot initiation and finally root initiation media forregeneration of transgenic plant.

Modulation of Regulatory Nucleic Acids

In further aspects, the invention provides methods for modulating theregulatory activity of MT3-A regulatory nucleic acids. Such modulationmay be achieved, for example, by administering to a plant or plantmaterial, a compound (or mixture of compounds) with modulatory activity,such as a small molecule (such as a plant hormone), protein, RNA, DNA,metal compound, oxidizing agent or virus. The following sectiondescribes various exemplary methods that may be used for identifyingsuch compounds.

In one embodiment, a compound can be identified by performing assays inwhich an MT3-A regulatory nucleic acid binding partner is incubated withsuch a nucleic acid and the effect of a test compound on the specificbinding of the binding partner to the nucleic acid is determined. Thebinding partner can be, for example, a nuclear extract prepared from acell expressing MT3-A, such as mesocarp cells or senescing leaf cells.Alternatively, the binding partner can be an isolated, purified orcloned transcription factor. Modulation of binding to the nucleic acidcan be determined, e.g., in an electrophoretic mobility shift assay(EMSA), such as those described above. Thus in this exemplary method, atest compound can be incubated together with a nucleic acid, which ispreferably labeled, comprising an MT3-A regulatory nucleic acid and thebinding partner. The reaction mixture is then subjected to anelectrophoresis and the amount of “retarded” protein-nucleic acidcomplex is compared to the amount of retarded complex from a bindingreaction in which the test compound has not been added. A lower level ofcomplex observed in the reaction that contains the test compoundcompared to the level of complex observed in the reaction that does notcontain the test compound indicates that the test compound inhibits orreduces binding of one or more binding partners to the MT3-A regulatorynucleic acid. This type of assay may be performed as a high-throughputassay.

Several in vivo methods may also be used to identify compounds thatmodulate an MT3-A regulatory nucleic acid. In one embodiment, theinvention provides a method comprising incubating a cell expressingMT3-A with a test compound and measuring the MT3-A mRNA or proteinlevel. mRNA levels can be determined by a variety of method known in theart including Northern blot hybridization, RT-PCR, microarray analysis,etc. Protein levels can be determined by a variety of method known inthe art including immunoprecipitations or immunohistochemistry using anantibody that specifically recognizes MT3-A.

In a further exemplary embodiment, a reporter construct can beconstructed in which a reporter gene is regulated by an MT3-A regulatorynucleic acid. The reporter gene can be any gene encoding a protein whichcan readily be detected. According to the method of the invention, cellsor plants are transformed with the reporter construct. The material canthen be incubated in the presence or absence of a test compound for anappropriate amount of time and the level of expression of the reportergene can be determined. Compounds which produce a statisticallysignificant change in expression of the reporter gene can be identified.

Plant Products

In certain aspects the invention provides products obtained fromtransgenic plants. Such products are intended to include any materialthat can be obtained from a plant, whether refined, purified, crude orsimply a part or a whole of the plant itself. Exemplary plant productsinclude fruits, juices, saps, syrups, oils, wood chips, nut meats,nectars, phytochemicals (chemicals extracted from plants), flours,proteins, enzymes etc. Plant products may refer to products that areproduced by a plant only because of a genetic manipulation. For example,transgenic plants may be designed to produce unusual, novel ornon-endogenous oils, novel or non-endogenous proteins, plastics such aspolyhydroxybutyrates, etc.

Often plant products will be sufficiently crude that there will bedetectable nucleic acids present. In such case, it is possible toascertain the plant from which the product was derived by analyzing thenucleic acids. For example, a crude extract from the mesocarp of an oilpalm fruit may contain nucleic acids (whether free or within remainingintact cells). In such case it is possible to detect the presence of anucleic acid of interest, such as an MT3-A regulatory nucleic acid, toobtain information about the source plant.

Oil may be extracted from palm tissues in a variety of methods known toone of skill in the art. In certain embodiments, crude palm oil isextracted from the oil palm mesocarp in palm oil mill using mechanicalscrew press method. An exemplary protocol, described in more detail byMa A N (1994) [“Extraction of crude palm oil and palm kernel oil.Selected Readings on Palm Oil and its Uses”. (Eds. Technical Committeeof 1994 Palm Oil Familiarization Programme). Palm Oil Research Instituteof Malaysia, 24-34] can be summarized as follows:

Ripe fresh fruit bunches (FFB) are harvested from oil palm plantationand transported with care to the mill to minimize damage. Damaged fruitswill result in poor oil quality due to increase levels of free fattyacids. Prior to oil extraction, the FFB are subjected to systematicpretreatments, which begin with sterilization, followed by stripping andfinally digestion. During sterilization, the FFB are placed insterilizer cages and subjected to steam-heat treatment at saturatedpressure of 3 kg/cm² and a temperature of 140° C. for 75 to 90 minutes.Fruit sterilization is performed for the following reasons:

-   -   i) to prevent increase in free fatty acid due to enzymatic        activities;    -   ii) to facilitate stripping of fruits from the spikelets;    -   iii) prepare the fruit mesocarp for digestion process.        Subsequently, the FFB undergo stripping process in a rotary drum        stripper where the fruits are separated from the spikelets. The        released fruits are then subjected to digestion process where        they are mashed under steam-heated conditions. This breaks the        oil-bearing cells of the mesocaip.

Unrefined oil is pressed out from the pretreated fruits under highpressure using twin screw presses. The crude oil flows into a tank forfurther purification. The pressure should be optimized to press out allthe oil from the mesocarp without breaking the kernel. The fibre and nut(press cake) are conveyed to a depericaper for separation. In somemills, double pressing is employed to minimize nut breakage. In thissystem, the fibre, after separation from the nut is sent for secondpressing to recover the residual oil.

Metallothionein Nucleic Acids and Proteins

In further aspects, the invention relates to metallothioneins, whichterm includes metallothionein-like proteins, and the nucleic acidsencoding such metallothioneins. Metallothioneins (MTs) are an extensiveand diverse family of small cysteine-rich proteins that are found in allorganisms. Their name derives from their high sulfur content and abilityto bind metals in stable metal-thiolate clusters. In view of theirmetal-binding capacity, it has been suggested that MTs may play a rolein the homeostasis of essential metal ions and the detoxification ofheavy metals, such as Cd²⁺ or Hg. However, MTs have now been implicatedin a wide range of biological processes relating to normal developmentand both biotic and abiotic responses. There are also numerousindications that MTs are involved in responses to oxidative stress,possibly by cavenging peroxyl and free hydroxyl radicals. Tissue anddevelopmental-specific expression of MT-like genes were demonstrated inseveral plant species such as Arabidopsis and kiwifruit suggestingpossible involvement in developmental processes.

In certain embodiments, the invention provides methods of generatingtransgenic plants comprising a heterologous nucleic acid encoding ametallothionein or a nucleic acid encoding a metallothionein that isexpressed from a heterologous promoter. In one embodiment, nucleic acidsencoding a metallothionein comprise nucleic acids that are at least 75%identical to one or more of the nucleic acids of SEQ ID Nos:2-4. SEQ IDNos: 2 and 3 are cDNA sequences. SEQ ID Nos: 4 is a genomic sequence. Inother embodiments, nucleic acids encoding a metallothionein comprisenucleic acids that are at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the nucleic acid of SEQ ID Nos:2-4. In a furtherembodiment, the nucleic acids encoding a metallothionein comprisenucleic acids that are a portion, or a functionally active assemblage ofseveral portions of the nucleic acid of SEQ ID No:2-4. It is furtherunderstood that nucleic acids may be interrupted by artificial introns,and it may be advantageous to position leader sequences and trailersequences, such as terminators, at the 5′ and 3′ ends, respectively. Inpreferred embodiments, nucleic acids encoding a metallothionein retainthe ability to interact with metals, and particularly transitionalmetals and/or d10 metals.

In certain aspects the invention provides nucleic acid constructscomprising a metallothionein nucleic acid as described above operablylinked to a promoter. The invention further provides vectors comprisingthe above nucleic acid constructs, and preferred vectors are suitablefor transformation of plants and/or plant cells and/or plant tissues.Metallothioneins may be expressed in plants using the normal promoterassociated with the gene, or different, i.e. heterologous, promoters. Aconstitutive promoter such as the CaMV 35S promoter may be selected, or,for example, the nopaline synthase promoter from Agrobacterium.Promoters having different properties, such as greater tissuespecificity may also be selected. In this manner it is possible todirect metallothionein expression to a variety of different, selectedtissues. It may be desirable to select a promoter from the same organismas the metallothionein is to be expressed in.

Accordingly, in certain aspects the invention provides transgenicplants, tissues and cells comprising a nucleic acid complex as describedabove. Such plants will typically have a higher level of the expressedmetallothionein protein present in transformed cells than in theuntransformed state. Such plants may also have a lower level ofuncomplexed metals.

The expression of metallothioneins in a tissue or a whole plant has ahost of desirable effects. Metallothioneins may help complex certainmetals and thereby increase the resistance of cells expressing themetallothionein to metal toxicities. Metallothioneins are alsoprotective against oxidative damage. Metallothioneins also scavenge freeradicals and are thus protective against such compounds.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXAMPLE Brief Summary of the Examples

Two differentially expressed type 3 MT-like genes were isolated from theoil palm and designated MT3-A and MT3-B. The presence of two copies oftype 3 MT-like genes in the oil palm genome was confirmed by Southernblot analysis. The coding sequence of MT3-A and MT3-B are 78% and 70%identical at the nucleotide and amino acid levels, respectively. Thehomologies within the 3′ and 5′-UTR were only 47% and 41%.

Northern analysis showed that both genes are expressed in the mesocarpthroughout the ripening period with maximum expression at 15 weeks afteranthesis (w.a.a) but MT3-A transcripts were 50 times more abundant thanthose of MT3-B. Their expression is highly selective to mesocarptissues. No detectable expression of MT3-A was found in kernel, roots,germinated seedlings and young leaves. Expression of MT3-B was notdetected in kernel, germinated seedlings and leaves. However, MT3-B isalso expressed at low levels in roots while the expression of MT3-A isinduced in senescing leaves.

The highly mesocarp-selective expression of MT3-A may have implicationsfor plant biotechnology generally, and in particular oil palmbiotechnology. For example it was found that the MT3-A expressionpattern is very similar to that of stearoyl-ACP desaturase, an enzymewith direct involvement in mesocarp oil synthesis. Since MT3-A is highlyexpressed in the mesocarp tissue of Elaeis guineensis, the high yieldingcommercial oil-bearing species, nucleic acids that regulate MT3-Aexpression would be valuable for expressing foreign genes aimed ateffecting oil modification in the mesocarp. This is a major goal ofefforts to improve oil palm crop. Of course, by using the entirepromoter of MT3-A, the transgenes would also be expressed in senescingleaves but this is unlikely to be detrimental to the plant as product ofthe introduced gene would be rapidly degraded as senescence proceeded.

The genome walking approach was used to clone the promoter of MT3-A.Primers from within the coding region of MT3-A were used to capture thepromoter or 5′ regulatory sequence. The overlapping region of the clonedPCR product showed total homology with MT3-A cDNA sequence. The promotersequence of MT3-B had been obtained earlier and submitted to the publicdata base (accession no. AJ236914). There is no significant homologybetween the promoter sequence of MT3-A and MT3-B. The specificity ofMT3-A promoter was confirmed by analysis using transient assay system.In this analysis, the oil palm MT3-A gene promoter was cloned into apromoterless transformation vector containing green fluorescence protein(GFP) as reporter gene and used in bombarding oil palm mesocarp slicesand control leaf tissues. Transient expression of GFP observed as greenfluorescence spots were detected in the mesocarp slices and not in thecontrol tissue. This result was confirmed using β-glucuronidase (GUS) asreporter gene in the transient assay analysis. Comparison was madebetween the activity of a constitutive cauliflower mosaic virus (CaMV)35S promoter and MT3-A promoter. Transient expression of GUS can bedetected on both mesocarp slices and control tissues bombarded with geneconstruct containing the constitutive promoter. Expression was onlyobserved on mesocarp slices bombarded with gene construct containingMT3-A promoter.

Example 1 Screening cDNA Library with Subtracted cDNA Probes

Methods

Biotin labeled mRNA was produced by mixing 10 μg of 5 w.a.a mesocarpmRNA with 30 μl of photoactivable biotin and irradiating the mixture onice with 300 W reflector flood lamp; for 20 min. The solution wasextracted with 2 volumes of water-saturated butanol and thebiotin-labeled mRNA recovered in the aqueous phase was ethanolprecipitated.

Single stranded cDNA was synthesized from 15 w.a.a mesocarp in 25 μlvolume containing 1.0 μg mRNA, 0.5 μl RNase inhibitor (10 U/:l), 5 μl of5× reverse transcriptase buffer, 1.25 μl of 80 mM sodium pyrophosphate,1.0 μl of 100 mM dNTP mix, 25 U reverse transcriptase at 42° C. for 1 h.The solution was phenol extracted, ethanol precipitated and the pelletdissolved in 30 μl H₂O.

The biotin-labeled 5 w.a.a. mRNA solution was added to the 15 w.a.a.cDNA solution, ethanol precipitated and dissolved in 10 μl H₂O and 10 μlof 2× hybridisation buffer (Stratagene). The mixture was incubated at100° C. followed by hybridisation at 68° C. for 48 h. A 30 μl volume of10 mM HEPES/EDTA buffer pH 7.5 and 10 μl streptavidin (1 mg/ml) wereadded and the tube was incubated at room temperature for 10 min.Phenol/chloroform extraction was performed. The aqueous phase containingsubtracted cDNA was removed and ethanol precipitated. Labelled probeswere prepared from the subtracted cDNA using random primed DNA labelingsystem from Stratagene.

Mesocarp cDNA library used for screening with the subtracted cDNA probeswere from 15 w.a.a tissues from E. guineensis constructed in Uni-ZAP-XRvector (Stratagene). Ten 110 mm petri dishes were used to plate about200,000 plaque forming units (pfu) for primary screening. The plaqueswere generated in E. coli host strain XL1-Blue. Preparation of cells forplating were prepared based on Sambrook et al. (1989) and plaque liftswere carried out as described in Siti Nor Akmar et al. (1995)[“Construction of oil palm mesocarp cDNA library and the isolation ofmesocarp-specific cDNA clones”. Asia Pacific Journal of MolecularBiology and Biotechnology, 3: No. 2: 106-111].

Hybridization of probes to membranes was carried out in hybridizationbuffer containing 5×SSPE (1×SSPE is 0.18 M NaCl, 10 mM NaH₂PO₄, pH 7.5,1 mM EDTA), 5×Denhardt's solution (1×Denhardt's solution is 0.02% eachFicoll 400, bovine serum albumin, and polyvinylpyrrolidone), 0.5% SDS,100 mg/ml denatured salmon sperm DNA, and 1×10⁶-5×10⁶ cpm/ml probes.After hybridization the membranes were washed twice in 0.1×SSPE, 0.1%SDS at 65° C. for 20 min. The membranes were subsequently exposed toX-ray films for 48 hr at −80° C.

Results

Biotinylated mRNA from very young mesocarp tissue at 5 weeks afteranthesis (w.a.a) was used in hybridisation with single strand cDNAmolecules derived from 15 w.a.a mesocarp tissue to remove commonsequences including housekeeping genes and other genes expressed in bothtissues. The coupled mRNA-cDNA hybrids were efficiently removed withstreptavidin. The remaining unhybridised cDNAs which were enriched withsequences specifically expressed in 15 w.a.a mesocarp tissue werelabeled and used as probes in screening a 15 w.a.a oil palm mesocarpcDNA library constructed in Uni-ZAP-XR vector (Stratagene). After thesecondary screening, five clones which produced strong hybridizationsignals with the subtractive probes but no signals with 5 w.a.a cDNAprobes used as a negative control were selected.

The inserts from all five clones cross-hybridised with each other andwere subsequently shown to be identical in sequence. The longest clonewas used in rescreening the 15 w.a.a mesocarp cDNA library to obtain afull length clone. About 1% of the recombinant clones from the libraryproduced hybridisation signals indicating that the probe corresponded toa very abundant gene in this tissue. The nucleotide and the deducedamino acid sequence of the biggest clone isolated was designated pOPSN6.pOPSN6 contains a 490 bp insert with an open reading frame (ORF) of 198bp (FIG. 1). Search for homologies with protein sequences in thedatabases using BLASTP selected exclusively metallothionein-like(MT-like) proteins and metallothioneins from various organisms as thetop 50 best matches. The greatest similarity of 77% identity, was withsequence from Musa acuminata (banana; [Q40256]) which belongs to thequite recently identified group of genes isolated from ripening fruit ofa few plant species referred to as type 3 MT-like genes. The encodedprotein of pOPSN6 appears to have the typical structure of plant MT-likeproteins with two cysteine-rich (Cys-rich) terminal domains separated bya cysteine-free (Cys-free) central domain as described by Robinson etal, (1993) [“Plant metallothioneins.” Biochem. J. 295:1-10]. Differenttypes (type 1, 2 and 3) of plant MT-like genes are identified bycharacteristic arrangement of cysteine residues of their encodedpolypeptides. FIG. 2 shows the amino acid sequence alignment of thededuced amino acid sequence of the oil palm gene and the sequences ofdifferent types of plant MT-like proteins. The oil palm sequence has thesame number of cysteine residues which are arranged in a specific andconserved pattern in two domains at the N and C terminals as the othertype 3 MT-like genes from different plant species confirming that itbelongs to this group of proteins.

Example 2 5′-RACE

Methods

First strand cDNA was synthesized from 15 w.a.a. oil palm mesocarp RNAusing antisense 3′ sequence specific primer MI (SEQ ID NO: 8) (5′ CTACCA ATA GCA ATC CAT TAA 3′) from 3′-UTR of pOPSN6 in a 20 μl reactionmixture containing 5 μg total RNA, 5.0 μl of 2 mM dNTP mix, 2.0 μl of0.1 M DTT, 1 μl of 200 U/μl Superscript reverse transcriptase (GibcoBRL) and 4 μl of 5× Superscript buffer at 42° C. for 1 hr. The RNAmolecules were hydrolysed in 12.5 μl of 0.15 N sodium hydroxide and 1μl, 0.5 M EDTA, pH 8.0 and incubated at 68° C. for 15 min. A poly(dG)tail sequence was introduced with terminal deoxynucleotidyl transferasein a 20 μl reaction mixture containing 10 mM Tris-acetate pH 7.5, 10 mMmagnesium acetate, 50 mM potassium acetate, 0.2 μl 100 mM dGTP and 0.2μl of 300 U/μl enzyme. Second strand cDNA was synthesized in a 50 μlreaction mixture containing 5.0 μl of 2 mM dNTP, 50 μmol of anchorprimer KAI (SEQ ID NO: 29) (5′ CU CCC CCC CCC CCC C 3′), 4.5 μl ofdG-tailed single-stranded cDNA, 2.6 U of Expand High Fidelity polymerase(Boehringer Mannheim) and 5 μl of 10× enzyme buffer containing 1.5 MMMgCl₂. PCR conditions were as follows: 1 cycle: 95° C. for 3 min, 43° C.for 1 min, 72° C. for 2.5 min, followed by 4 cycles; 95° C. for 1 min,43° C. for 1 min, 72° C. for 2 min.

The PCR product was purified using QIAquick PCR purification kit(Qiagen), and eluted using 50 μlμH₂O. A 5 μl aliquot was added to 50 μlof a secondary PCR mixture containing 5 μl of 2 mM dNTP, 50 pmol ofnested primer M32 (SEQ ID NO: 10) (5′ CAC CAT GAC AGA AAC ATA TC 3′),2.6 U of Expand high fidelity polymerase and 1× enzyme buffer containing1.5 mM MgCl2. The following PCR conditions were used: 1 cycle; 95° C.for 3 min, 51° C. for 1 min. 72° C. for 30 sec, followed by 9 cycles;95° C. for 1 min, 51° C. for 1 min, 72° C. for 1 min and 30 sec, and 80°C. soak during which 50 pmol of anchor primer KA1 was added followed by30 cycles; 95° C. for 1 min, 51° C. for 1 min and 72° C. for a min. ThePCR product was purified and cloned into PCRII-TOPO vector (Invitrogen).

Results

Total RNA from 15 w.a.a. oil palm mesocarp was used as template in 5′RACE reactions in order to obtain the full length cDNA sequence and todetermine the transcription start site of this gene. First strand cDNAsynthesis was carried out using primer M1 based on the sequence at the3′ end of the pOPSN6 just prior to the poly(A) tail. Homopolymer tailpoly (dG) was subsequently introduced using terminal transferase. Secondstrand cDNA was synthesized using poly(dC) anchor primer KA1. The anchorprimer KA1 together with nested primer based on internal 3′ sequence ofpOPSN6 were used in the secondary PCR reaction to ensure amplificationof the desired sequence. The product from the secondary PCR reaction wascloned and sequenced. The sequence obtained as shown in FIG. 1 b showedfurther 5′ extension to the original cDNA sequence of pOPSN6.

Example 3 Screening of Genomic Library

Methods

Ten 110 min petri dishes were used to plate about 200,000 plaque formingunits (p.f.u) for primary screening. The plaques were generated in E.coli host strain XL1-Blue MRA(P2) Preparation of cells for plating wereprepared based on Sambrook et al. (1989) and plaque lifts were carriedout as described in Siti Nor Akmar et al. (1995).

Results

About 200,000 clones from an oil palm genomic library constructed inlambda FIX II (Stratagene) were screened using random prime-labeled PCRproduced probe containing pOPSN6 sequence. The strongest hybridisingphage was isolated and the DNA purified.

Example 4 Southern Blot Analysis and Identification of MT3-B

Methods

Large scale bacteriophage lambda DNA from clones isolated from thegenomic library was prepared using the plate lysate method as describedin Sambrook et al., (1989). Digestion with restriction enzymes wascarried out according to manufacturer's instructions. Electrophoresis ofdigested samples was performed using 0.9% agarose gel in TAE buffer (40mM Tris-acetate pH 7.9, 1 mM EDTA). Following electrophoresis, the gelwas gently shaken in 0.25 M HCl for 10 min for depurination prior totransfer. The DNA was denatured during overnight transfer onto Hybond N⁺(Amersham) membrane under alkaline conditions using 0.4 M NaOH as thetransfer buffer. The membrane was rinsed in 2×SSPE before continuingwith prehybridization.

PCR produced probes were labeled using Megaprime DNA labeling systemfrom Amersham. Hybridization was performed as described above. Formedium stringency washes, the membrane was washed twice in 4×SSPE and0.1×SDS at 50° C. for 15 mins. The membrane was exposed to Kodak X-OMATX-ray film for 1 week.

Results

Southern analysis was carried out using various restriction enzymes withpOPSN6 as probe and the appropriate fragment was selected and clonedinto pBluescript SK−. This fragment was further digested to produce 6fragments for subcloning to assist in sequencing.

Example 5 Sequence Analysis

Methods

Plasmid DNA for sequencing was extracted using the Qiagen plasmid minikit. DNA sequencing was carried out from both directions using ABIautomated sequencer. The DNASIS Sequence Analysis Software was used forsequence analysis and search for similarity between nucleotide and aminoacid sequences.

Results

Sequence obtained using M13 reverse primer from one end of one of thesubclones picked up homology with amino acid sequence at the C-terminaldomains of the encoded proteins of plant MT-like genes. Oil palmmesocarp mRNA was used as template in 5′ RACE reactions in order toconfirm the coding region within the genomic sequence and to determinethe transcription start site of this gene. A total of 10 cDNA clonesfrom two independent RACE reactions were sequenced. Eight of theseclones had identical sequence and one of them designated pOPSN7, whichcontained a 399 bp insert provided the longest sequence at the 5′ end.

The sequence of pOPSN7 contains a 192 bp ORF coding for 63 amino acidswith predicted molecular weight of 6.5 kDa. The sequence contains 76 bpof 5′-UTR. The deduced amino acid sequence of pOPSN7 was found to bedifferent from pOPSN6 but it has the characteristic arrangement ofcysteine residues as other plant type 3 MT-like genes. Therefore it wasconcluded that the genomic clone isolated corresponded to another type 3MT-like gene from the oil palm which is different from pOPSN6. The twooil palm type 3 MT-like genes were thus designated MT3-A (pOPSN6) andMT3-B (pOPSN7).

Strongly homologous nucleotide sequences between pOPSN6 and pOPSN7 weremainly found in the coding region which has 78% sequence identitity(FIG. 3). The homologies within the 3′ and 5′-UTR were only 47% and 41%.The identity at the amino acid level was 70% and the homology increasedto 86% if conservative substitutions were considered. In addition to thecysteine residues, the first 20 residues from the N-terminal whichincludes five charged residues (two Ds and three Ks) are highlyconserved. The central and C-terminal contain a total of three conservedcharged residues (two Es and one K).

A 3.9 kb genomic sequence of MT3-B was obtained and submitted to theEMBL database and was given the accession number AJ236914. The sequencehas 2 AT-rich (about 64% AT) introns of 1.432 kb and 306 bp. The intronsdivide the coding region into 3 exons of 47 bp, 48 bp and 97 bp. Thegenomic sequence also contains 763 bp of 5′ and 1.243 kb of 3′ flankingregions.

Genomic Southern analysis was carried out to determine the gene copynumber of the oil palm MT3-A and MT3-B (FIG. 4) based on the methoddescribed in Example 4. Gene-specific PCR produced probes based on the3′-UTRs of these genes hybridised to a single fragment in restricted oilpalm genomic DNA even with low stringency washing conditions indicatingthat there is probably only one copy of each gene in the oil palmgenome. Oil palm genomic DNA was extracted based on the method ofDellaporta et al. in [“A plant minipreparation: version II. Plant Mol.Biol. Report 1: 19-21]. Southern analysis has also been performed usingthe entire pOPSN6 insert as probe (data not shown). At low stringencyconditions, the probe hybridised to two fragments in restricted oil palmgenomic DNA but one of them was much fainter than the other. It waslater found that the faint band migrated the same distance as the MT3-Bhybridising fragment. This further confirms the presence of two MT3genes in the oil palm genome which are MT3-A and MT3-B.

Example 6 Northern Blot Analysis

Methods

Different tissues from E. guineensis, tenera variety, were used. Freshfruit bunches were harvested at various weeks after anthesis (w.a.a.)for RNA extraction from the mesocarp and kernel tissues. Young andsenescing leaves from 3-4 months polybag seedlings were obtained for RNApreparations. Germinated seedlings after 1 week undergoing germinationprocess were used for RNA extraction.

Total RNA was extracted from various oil palm tissues as describe inSiti Nor Akmar et al. (1994 Detection of differentially expressed genesin the development of oil palm mesocarp. Asia Pacific Journal ofMolecular Biology and Biotechnology, 2: No. 2, 113-118). Messenger RNAsfrom various oil palm tissues were prepared as described in Siti NorAkmar et al (1994). Two microlitres of mRNA (0.5 μg/μl) was denatured in18 μl L of solution containing 78% (v/v) deionized formamide, 16%deionized glyoxal, and 10 mM NaH₂PO₄/Na₂HPO₄ (pH 7.0) by heating for 15min at 55° C. followed by immediate cooling. Denatured mRNA wasseparated on 1.2% agarose gel using 40 mM Tris-acetate (pH 7.0) aselectrophoresis buffer. Transfer to nylon membrane (Hybond-N Amersham)was carried out using a vacuum blotter (60 cm H₂O, 4 hr) in 20×SSC(1×SSC is 0.15 M NaCl, 15 mM trisodium citrate 2H₂O, pH7.0).Hybridization and preparation of labeled probes were carried out asdescribed above.

Results

Northern blot analysis was carried out to determine the expressionpatterns of the oil palm type 3 MT-like genes in various oil palmtissues. Initial analysis was carried out using the entire insert ofpOPSN6 (MT3-A cDNA clone) as a probe. Poly (A)⁺ RNA from mesocarp atdifferent stages of fruit development, from very young (5 w.a.a) to ripefruits (20 w.a.a) as well as from kernel at 12 w.a.a were used. Poly(A)⁺ RNA from vegetative tissues (young leaves and germinated seedlings)were also included in the analysis.

It was observed that the MT3-A probe hybridised to a single transcriptof about 530 bp (FIG. 5). The expression of MT3-A in the mesocarptissues was very high and developmentally regulated. MT3-A transcriptswere not detectable in the mesocarp at 5 w.a.a. MT3-A gene expressionwas already high at 12 w.a.a, highest at 15 w.a.a and decreasingsignificantly as the fruits ripened. The expression was not detectablein kernel at 12 w.a.a and young leaves while a trace level was observedin germinated seedlings. From this analysis the expression of MT3-Aappeared to be very strong and specific to the mesocarp.

Northern blot analysis was carried out to compare the expressionpatterns of the two different type 3 oil palm MT-like genes (MT3-A andMT3-B) using gene-specific probes based on their 3′-UTR. The same probeswere used for Southern analysis as described above. Total RNA fromdifferent developmental stages of mesocarp and kernel as well as fromroots, leaves and senescing leaves were used. Both probes hybridised tosimilar size transcripts of about 530 bp (FIG. 6). The expressionpattern of MT3-A was found to be different from MT3-B. MT3-A expressionin the mesocarp can be detected as early as 8 w.a.a., reached a maximumat 15 w.a.a. and decreasing significantly as the fruits ripened. Tracelevels of expression was detected in roots and kernel at 15 w.a.a. Theexpression was not detected in young leaves but was induced in senescingleaves.

The expression of MT3-B in the mesocarp was shown to be 50-fold lowerthan MT3-A and more specific to the ripening stage. The expression wasnot detectable in young mesocarp tissue of 8 w.a.a, trace level wasobserved at 12 w.a.a, highest at 15 w.a.a and reducing slightly at 17w.a.a. The presence of MT3-B transcripts in 12-weeks mesocarp tissue wasconfirmed by detection of signals with longer exposure time of the X-rayfilm. The expression in roots was also high and the level was similar tothat observed in 17 w.a.a mesocarp. MT3-B gene transcripts were notdetected in kernel, leaves and senescing leaves.

FIG. 7 shows the expression pattern of two different oil palm genesencoding stearoyl-ACP desaturase (SAD), an enzyme directly involved inoil synthesis in the mesocarp tissues. SAD is responsible for convertingsaturated stearoyl-ACP to monounsaturated oleoyl-ACP. Constitutiveexpression of SAD2 suggests a possible housekeeping role in membranelipid biosynthesis. We were more interested in comparing the expressionpattern of MT3-A with that of SAD1. SAD1 is believed to have a directinvolvement in storage oil synthesis because its expression is inducedin lipid-rich mesocarp and kernel tissues in phase with oil synthesis.SAD1 however, may also play a role in membrane lipid synthesis inactively dividing young tissues because it is also quite highlyexpressed at 8 w.a.a. Similar to SAD1, MT3-A are expressed throughoutthe ripening period in the mesocarp with an expression peak at 15 w.a.aMT3-A transcripts however, start increasing earlier at around 8 w.a.aand its expression is also induced in senescing leaves.

Example 7 Promoter Isolation

Methods

Isolation of the mesocarp-specific promoter was carried out using theUniversal Genome Walker Kit (Clontech). Total DNA was isolated andpurified from oil palm spear leaves using DNeasy Plant Mini Kit fromQiagen. Aliquots containing 2.5 μg DNA were digested with restrictionenzymes Dra I, Eco RV, Pvu II and Stu I that produce blunt ends andligated to the GenomeWalker Adaptor creatine the GenomeWalker libraries.Primary PCR was performed using 1 μl aliquots of each library withantisense gene-specific primer GSP1 (SEQ ID NO: 11) 5′CCACACAAGCACAGCTAGCACCACACTTG 3′ from 3′-terminal of the coding regionof 15 pOPSN6 and primer API provided with the Kit. The PCR product wasdiluted 50× and 1 μl was used in secondary PCR reaction using antisensenested gene-specific primer GSP2 (SEQ ID NO: 12) 5′CTGGCTCTTGTCAGCACAATCGCAGTTGC 3′ from the 5′-terminal of pOPSN6 codingregion and primer AP2 front the Kit. PCR was carried out using AdvantageTth Polymerase Mix front Clontech and Perkin-Elmer 9600 thermal cyclerfollowing cycle conditions recommended in the GenomeWalker Kit Manual.The secondary PCR product was analysed and purified from agarose gelusing gel extraction kit front Qiagen and cloned into PCRII-TOPO vector(Invitrogen). The recombinant clone was sequenced using M 13 forward andreverse primers.

Results

The Universal GenomeWalker kit from Clontech was used to clone thepromoter or the 5′ upstream regulatory region of MT3-A. Separatealiquots containing 2.5 □g DNA were digested with four differentrestriction enzymes namely Dra 1, Eco RV, Pvu II and Stu I whichproduced blunt ends. The GenomeWalker Adaptor was subsequently ligatedto the restriction fragments creating four different GenomeWalkerlibraries. Aliquots from these libraries were amplified using a 29-mergene-specific primer (GSP1) and primer AP1 from the Adaptor sequence.GSP1 was designed based on the sequence near the 3′-end of MT3-A codingregion (bases number 246 until 275 in FIG. 1). GSP1 sequence fallswithin a fairly variable region of plant type 3 MT-like gene sequences.In this region, 3 out of 8 amino acid residues of MT3-A are differentfrom MT3-B. The size of bands from this primary PCR reaction is shown inFIG. 8A. The Dra L Eco RV and Stu I libraries produced PCR products ofabout 1.2 kb, 1.0 kb and 0.45 kb, respectively. Since the product of theDra I library was the biggest, it was selected for further PCR reaction.One microliter of 1/50 dilution of the primary PCR product was used in asecond round PCR reaction. In this reaction a 29-mer nestedgene-specific primer (GSP2) and nested primer from the Adaptor sequence(AP2) were used. GSP2 was designed based on the sequence at the 5′-endof MT3-A coding region (bases number 111 until 139 in FIG. 1). Thissecondary PCR reaction specifically amplified fragments containing MT3-Asequence. Fragments produced in the primary PCR reaction due tonon-specific binding of primers were not amplified. FIG. 8B showed theproduct of the secondary PCR reaction. The size of band obtained wasapproximately 1.2 kb but slightly smaller than the primary PCR product.This is the expected size using the pair of nested primers because thesequence of GSP2 is only 70 bp internal to GSP1. This may also suggestthe absence of introns in the genomic sequence. If an intron is presentwe will expect the secondary PCR product to be smaller. The band waspurified from the agarose gel and cloned into PCR II TOPO vector(Invitrogen). Two of the recombinant clones designated pMT3A-P1a andpMT3A-P1b were sequenced from both directions using M13 forward andreverse primer. The sequences of both clones were found to be identical.

The complete sequence of pMT3A-P1a is given in FIG. 9. The sequence hasno significant homology with the promoter sequence of MT3-B. A putativeTATA box and an ethylene responsive element in reverse orientation(ERE-reverse) were identified at position 953 and 669. The ERE issimilar to the ERE AATTCAAA of ripening-specific E4 gene of tomato ofMontgomery et al. (1993) [“Identification of an ethylene-responsiveregion in the promoter of a fruit ripening gene”. Proc. Natl. Acad. Sci.USA 90: 5939-5943] and the ERE ATTTCAAA associated with the regulationof carnation glutathione-S-transferase gene (GS7) in senescing tissues.The region containing the ERE in the GST1 gene was shown to operate inorientation—independent manner as described in Itzhaki et al., (1994)“An ethylene-responsive enhancer element is involved in thesenescence-related expression of the carnation glutathione-S-transferase(GSTI) gene”. Proc. Natl. Acad. Sci. USA 91: 8925-8929].

The sequence of pMT3-AP1a was aligned with the cDNA sequence of the 5′RACE product (FIG. 10). It was found that 113 bp of the 3′ terminalregion of pMT3A-P1a overlaps with the 5′-terminal sequence of the 5′RACE product. This contains 42 bp of coding sequence. Within theoverlapping region, the two sequences are identical. The putativetranscription start site is an denine that is 26 bp downstream of theTATA box (FIG. 9), which is consistent with the expected distance of32±7 (Joshi, C P 1987 An inspection of the domain between putative TATAbox and translation start site in 79 plant genes. Nucl. Acids Res. 15,No. 16: 6643-6653). In most genes the transcription start begins with anadenine.

Example 8 Cloning of the MT3-A Promoter into pEGFP-1 and pBI221

Methods

Two primers were used for cloning a 1040 by genomic fragment containing986 by MT3-A 10 promoter sequence and a further 54 bp sequencesdownstream into the multiple cloning site of pEGFP-1 (Clontech), apromoterless vector with GFP as reporter gene to produce MT3AP-EGFP. Thefirst primer gC3 (SEQ ID NO: 13) 5′ CCC AAG CTT AAA TTA CTG CCA TG 3′ isa sense primer front 5′ end of the promoter with an Hind III siteintroduced. The second primer gC4 (SEQ ID NO: 14) 5′ AAA ACT GCA GCA GGAAAC CAG AGA C 3′ is an antisense primer 16 bases upstream of thetranslation start site with a Pst I site introduced.

pBI221 plasmid (Clontech) contains β-glucuronidase (GUS) coding regionunder the control of a strong 35S promoter from cauliflower mosaic virus(CaMV). Plasmid MT3AP-GUS was produced by replacing CaMV 35S promoter inpB1221 with the oil palm MT3-A promoter. The CaMV 35S promoter wasremoved by digesting pB1221 with Hind III and Xba I. MT3-A promoter wasamplified using, primers gC3 and gC5. Primer gC5 (SEQ ID NO: 15) 5′ TGCTCT AGA CAG GAA ACC AGA GAC 3′ is similar to gC4 but in gC5, the Pst Isite is replaced with an Xba I site.

The PCR reaction mixture (50 μl) for amplifying MT3-A promoter contained5.0 μl of 2 mM DNTP, 3.3 μl of 15 μM gC3 primer, 3.3 μl of 15 μM gC4 orgC5 primer, 25 ng of plasmid pMT3A-P1a, 5.0 μl of 10× enzyme buffercontaining 1.5 mM MgCl₂ and 2.6 U Expand High Fidelity Polymerase(Roche). PCR conditions were as follows: 1 cycle; 94° C. for 3 min, 20cycles; 94° C. for 1 min, 42° C. for 1 min and 72° C. for 90 secfollowed by 1 cycle; 72° C. for 10 min. The PCR product was purifiedusing QIAquick PCR purification kit (Qiagen). Ligation was performedusing 1:3 molar ratio of vector:insert in 15.0 μl reaction volumecontaining 1.5 μl 10× ligase buffer and 1.5 μl of T4 DNA ligase (1 U/μl)and incubation at 16° C. O/N. Two microlitres were used to transformcompetent cells JM101 as described in Siti Nor Akmar (1999 Structure andregulation of stearoyl-ACP desaturase and metallothionein-like genes indeveloping fruits of the oil palm (Elaeis guineensis) PhD thesis,University of East Anglia, UK). The cells were spread on LB platecontaining 40 μl of 20 mg/ml5-bromo-4-chloro-3-indolyl-μ-D-galactopyranoside (X-gal and 40 μl of 20mg/ml isopropyl μ-D-thiogalactopyranoside (IPTG)) for blue/whiteselection of recombinant clones. Plasmid DNA was prepared from selectedwhite colonies using QIAprep spin miniprep kit (Qiagen). Restrictionanalysis was carried out by digesting with Hind III and Pst I forMT3AP-EGFP or Hind III and Xba I for MT3AP-GUS to confirm the size ofinsert. Sequencing of MT3AP-EGFP was carried out using EGFP-N sequencingprimer from Clontech. The successful replacement of 35S CaMV promoter bythe oil palm MT3-A promoter in pBI221 was verified by sequencing withM13 reverse primer.

Results

The 1040 genomic fragment containing 986 bp of mesocarp-specificpromoter sequence was cloned into a promoterless transformation vectorpEGFP (Clontech) containing GFP as reporter gene and the chimerictransformation vector produced was designated MT3AP-EGFP. Restrictionanalysis of the MT3AP-EGFP showed the successful cloning of the 1040 bpgenomic fragment in pEGFP (FIG. 11 a). The approximately 850 bp CaMV 35Spromoter was removed from pBI221 by digesting with Hind III and Xba I.The oil palm MT3-A promoter was flanked with Hind III and Xba I sitesand ligated to the digested vector and cloned into E. coli host.Restriction analysis of the cloned product MT3AP-GUS with Hind III andXba I showed the successful cloning of the 1040 bp genomic fragment(FIG. 11 b). The successful replacement of 35S CaMV promoter by the oilpalm MT3-A promoter in pBI221 was verified by sequencing with M13reverse primer.

Example 9 Biolistic Method and Transient Expression for PromoterAnalysis

Methods

Preparation of Tissue Slices

Oil palm fruits (12 w.a.a) were sterilized by soaking in between 20 for10 minutes followed by 25% chlorox for 20 minutes. The fruits were thenrinsed several times with sterile distilled water.

The sterilised fruits were cut into small pieces (1 cm×1 cm). Theexplants were put on culture media. These cultures were kept at 28° C.in the dark for 24-48 hours before bombardment.

The culture media were based on Murashige and Skoog (MS) salts. MS saltsand vitamins were commercially available in dried powder, from DUCHEFA,Biochemicals Plant Cell and Tissue Culture, Haarlem, Netherlands. Everylitre of media was prepared by dissolving 4.7 g MS salts and 30 gsucrose in distilled water. Media were solidified with 0.8%(w/v) agar.Media were adjusted to pH 5.8 with 1M NaOH solutions.

Bombardment Procedure

The oil palm fruits and leaf tissues were bombarded with Biolistic GunHe/100 (Biorad) U.S.A

-   -   The chamber (PDS-1000/He) was sterilised with 100% ethanol.    -   Microcarrier and macrocarrier were sterilised with 100% ethanol    -   a. Deagglomeration        -   60 mg of dry gold [microcarrier (1.0 μm in size)] were            placed in 1 ml of 100% ethanol in microcentrifuge tube            followed by vortexing for 1-2 minutes at 12,000 rpm. This            procedure was repeated 3 times. The mixture was sonicated            and the supernatant was removed before adding 1 ml of            sterile distilled water. The mixture was re-suspended,            centrifuged and finally the supernatant was removed.    -   b. Precipitation of DNA        -   Two of the plasmids used, HBT1α and MT3AP-EGFP contain green            fluorescence protein (GFP) as the reporter gene. HBT1α            contains 35S enhancer fused to the basal promoter of maize            C4PPDK gene (Chiu et al., 1996 Engineered GFP as a vital            reporter in plants. Current Biology, 6: No. 3: 325-330).            MT3AP-EGFP contains oil palm MT3-A promoter. Promoter            analysis was also carried out using β-glucuronidase (GUS) as            the repoter gene using constructed plasmid MT3AP-GUS. In            MT3AP-GUS, the CaMV 35S promoter in pBI221 was replaced with            the oil palm MT3-A promoter. All the plasmids were isolated            using QIAgen Spin Miniprep Kit. 30 μg DNA were added to 100            μl aliquot of gold. 100 μl 2.5M CaCl₂, 40 μl 0.1M spermidine            were added while vortexing. The mixture was centrifuged down            at 10,000 rpm. The supernatant, was removed and the            microcarrier was washed with 100% ethanol. These steps were            repeated twice and finally, the microcarrier was resuspended            in 60 μl ethanol and kept at −20° C. until used.    -   c. Operation        -   5-10 μl of DNA-coated microcarrier were placed onto the            centre of the macrocarrier. The mesocarp tissues were            bombarded with 1550 Psi helium pressure and 9 cm distance            between macrocarrier and target tissue, with 8 μl            gold-coated DNA loaded per bombardment. The leaves (control            tissues) were bombarded at 1100 Psi helium pressure and 6 cm            distance between macrocarrier and target tissue. The            distance between rupture disk to macrocarrier was fixed at 6            mm while the distance between macrocarrier to the stopping            plate was fixed at 11 mm. The vacuum pressure was maintained            at 27″ Hg.            GFP Expression.

The GFP expression was determined using Leica fluorescence microscopefitted with GFP filter set by counting the green fluorescence spotsproduced.

Results

Transient assay system was developed using biolistic method and mesocarptissue slices for analyzing the strength and confirming the specificityof oil palm mesocarp-specific promoter. In this method regions of thepromoter are ligated upstream to sequence encoding reporter genes suchas β-glucuronidase (GUS) and the green fluorescence protein (GFP).Tissue slices bombarded with the promoter:reporter gene construct willbe assayed for transient expression of the reporter gene. FIG. 12 givesthe results of an optimization experiment to determine the heliumpressure and the distance between the macrocarrier and taget tissuessuitable for bombarding the mesocarp slices and leaf tissues used asnegative control. The optimization experiment was carried out using theplasmid HBTla containing CAMV 35S enhancer and basal promoter of themaize C4PPDK gene (Chiu et al., 1996). In HBT1α, the a representsSGFP-nos.

Mesocarp tissue slices and leaf tissues were bombarded with MT3AP-EGFPusing the optimized bombardment parameters. Green fluorescenceindicating expression of GFP was observed on bombarded mesocarp slicesbut not on bombarded leaf tissues (FIG. 13). This confirms that MT3-Apromoter is functional and that it has mesocarp-specific activity.

Histochemical Assay for GUS

The substrate used for histochemical localization of β-glucuronidaseactivity was 5-bromo-4-chloro-3-indolyl glucuronide (X-glue). Thissubstrate works very well, giving a blue precipitate at the site ofenzyme activity. The bombarded oil palm tissues were fixed for 5 minuteson ice in fixation solution containing 5% formaldehyde in sodiumphosphate buffer pH 7.0. The histochemical assay was performed based onthe method of Jefferson et al. (1987) GUS fusions: glucuronidase as asensitive and versatile gene fusion marker in higher plants, EMBO J6(13): 3091-3097. The fixed tissues was transferred to X-GLUC solutionand incubated at 37° C. for several hours before analysis using a lightmicroscope.

-   -   X-GLUC solution        -   0.2M Na₂HPO₄        -   0.2M Na₂H₂PO₄        -   10% Triton X-100        -   X-gluc 50 mg/ml        -   0.25M Na₂EDTA        -   0.005M K Fe²⁺        -   0.005M KFe³⁺            Results

pBI221 is designed to express β-glucuronidase (GUS) fom the strongconstitutive CaMV 35S promoter. In order to compare the expression ofGUS under the control of the CaMV 35S promoter and the oil palm MT3-Apromoter, MT3AP-GUS was produced by replacing the CaMV 35S promoter inpBI221 with MT3-A promoter. pBI221 and MT3AP-GUS were used forbombarding oil palm mesocarp slices at 12 w.a.a. and leaf tissues.Histochemical assay of GUS activity was performed on the bombardedtissues. GUS expression was observed on both mesocarp slices and leaftissues bombarded with pBI221 (FIG. 14). Expression of GUS was onlydetected on mesocarp slices bombarded with MT3AP-GUS and no expressionwas observed on leaf tissues bombarded with this construct. These datafurther support the results obtained with GFP as the reporter gene (FIG.13), as it clearly indicates that MT3-A promoter is a functionalpromoter with mesocarp-specific activity.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated nucleic acid comprising a regulatory nucleic acid comprising the nucleic acid set forth in SEQ ID No: 1, or fragments of SEQ ID No: 1 with tissue specific promoter activity effective to regulate expression of an operably linked gene in the mesocarp.
 2. A transgenic oil palm comprising a recombined nucleic acid that is selectively expressed in the mesocarp, wherein expression of the recombined nucleic acid is regulated by an MT3-A regulatory nucleic acid, and wherein the MT3-A regulatory nucleic acid comprises SEQ ID NO:1.
 3. The transgenic oil palm of claim 2, wherein the recombined nucleic acid is also selectively expressed in one or more senescent leaves.
 4. The transgenic oil palm of claim 2, wherein the recombined nucleic acid is selectively expressed in the mesocarp during the period of oil synthesis.
 5. The transgenic oil palm of claim 2, wherein the recombined gene encodes a protein selected from the group consisting of: a phospholipase, a desaturase, a thioesterase, a thiolase, a hydroxylase, an oxidase, an acyl transferase, a synthase, a fluorescent protein, a reporter protein and a transcriptional regulator.
 6. The transgenic oil palm of claim 2, wherein the recombined gene encodes a protein that is involved in the metabolism of a compound selected from the group consisting of: a lipid, a sterol, a tocopherol, a carotenoid, a bioplastic, a tocotrienol and squalene.
 7. A method of obtaining an oil product comprising: (a) providing oil-bearing fruit from the transgenic oil palm of claim 2; (b) extracting oil from the mesocarp of the fruit.
 8. A method of claim 7 wherein the oil-bearing fruit is selected from the group consisting of: an oil palm fruit, an olive and an avocado.
 9. A method of claim 7 further comprising the step of enriching for one or more desired lipid. 